Hypoosmotic Solutions for Lymph Node Detection

ABSTRACT

Provided are compositions for rapid detection of lymph nodes. The compositions include magnetic particles, such as iron oxide, and a solute present in an amount that results in a hypoosomotic solution. Methods for detecting lymph nodes also are provided.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/202,178, filed on Mar. 10, 2014, which claims priority to U.S.Provisional Patent Application 61/775,780 filed Mar. 11, 2013, theentire contents of each of which are herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the field of medical diagnostics in general anddiagnostic methods and devices for locating tissue for surgicalexcision.

BACKGROUND

Approximately 1.25 million new cases of breast cancer are diagnosed eachyear. In a majority of these cases, there is an urgent need for surgeryto remove the tumor and to excise the sentinel lymph nodes and inspectthem histologically to determine whether the cancer has spread to othersites in the body. The sentinel lymph nodes are the first nodes toreceive lymphatic drainage from the tumor. They are called this becausethey reliably alert the clinician to any cancer spread. A sentinel lymphnode biopsy is a standard of care in breast cancer operations today.

Locating sentinel nodes during surgery is difficult. One method forlocating the sentinel node is to inject a dark blue dye into thelymphatic system in the breast. The dye then disperses throughout thebreast lymphatic system and the surgeon removes any colored nodes. Thismethod is recognized as being error-prone.

An improved method involves injecting a radioactive dye into the lymphnodes. In a similar manner, the dye drains through the lymphatic systemand the surgeon then uses a radiation detector to help locate thesentinel nodes. However, the use of radioisotopes presents asignificant, and an expensive, logistical burden, because of the need toallocate the time and resources of a nuclear medicine radiologist inaddition to the surgeon for what is otherwise a routine operation.Further, many patients are reluctant to receive a radioactive injection.These factors can become a significant barrier to the use ofradioisotopes to locate the sentinel nodes.

A further improved method involved injecting suspensions of magneticparticles into the lymph nodes and waiting for the magnetic particles todrain though the lymphatic system. The particles are then detected usinga magnetometer, which reveals the location of the lymph nodes. SeeUS2011/0133730. Prior art solutions, such as Sienna+®, have a very lowosmolality of about 30 mOsm/kg. Sienna+® is an aqueous solution ofmaghemite nanoparticles coated in carboxydextran, having an ironconcentration of about 25.5 to 29.5 mg/mL. It takes about 30 minutes forthe magnetic particles in a Sienna+® injection to drain sufficientlythrough the lymphatic system to ensure accurate lymph node detection,which can potentially cause significant and costly downtime duringsurgical procedures. Consequently, impatient physicians may attempt todetect the lymph nodes too soon—i.e., before the magnetic particles havesufficiently drained through the lymphatic system—which could result inincomplete lymph node detection.

A need therefore exists for compositions that enable more efficientprocedures.

The present invention addresses this need.

SUMMARY OF THE INVENTION

The invention relates to a hypoosmotic suspension for medical injection.In one embodiment, the composition includes about 13 mg/mL to about 200mg/mL of magnetic particles, and an osmolyte from either about 0.01% w/vto about 0.6% w/v of an inorganic salt (e.g., sodium chloride) or about0.5% w/v to about 1.5% w/v of a glycol.

Embodiments of the hypoosmotic suspensions can include one or more ofthe following features:

The magnetic particles can be iron oxide particles, such assuperparamagetic iron oxide particles (e.g., maghemite).

The magnetic particles can be coated, such as with dextran (e.g.carboxydextran).

The suspensions can have about 13 mg/mL of magnetic particles, about 28mg/mL of magnetic particles, 56 mg/ml of magnetic particles, 100 mg/mlof magnetic particles, 140 mg/ml of magnetic particles or about 200mg/mL of magnetic particles.

The suspension can have an osmolality of about 80 mOsm/kg to about 160mOsm/kg.

The suspension can include an excipient.

The inorganic salt can be present in the amount of about 0.01% w/v-0.6%w/v , about 0.05% w/v-0.3% w/v, about 0.1% w/v-0.3% w/v, less than about0.6% w/v or about less than about 0.3% w/v.

The invention also provides a method of locating a lymph node in apatient (e.g., a human). The method includes the steps of: providing ahypoosmotic suspension; injecting the hypoosmotic suspension into thepatient; waiting until the magnetic particles become entrapped in alymph node; and detecting the location of the lymph node by detectingthe location of the magnetic particles.

The method can include one or more of the following features:

The method can include injecting 0.2 mL of hypoosmotic suspension, 0.4mL of hypoosmotic suspension, or 0.8 mL of hypoosmotic suspension intothe patient.

The detecting can be performed using a magnetometer.

The invention also provides a method of rapidly locating a lymph node ina patient (e.g., a human). The method can include the steps of:providing a hypoosmotic suspension comprising magnetic particles;injecting the hypoosmotic suspension into the patient; and detecting alymph node within 10 minutes, or within as little as 5 minutes, ofinjection by detecting the location of the magnetic particles, thedetecting sufficient to immediately begin a medical procedure on thelymph node based on the detecting.

The invention also provides a method of treating a patient usingmagnetic hyperthermia, the method comprising the steps of: providing thehypoosmotic suspension; injecting the hypoosmotic suspension into thepatient; and exposing the patient to an alternating magnetic field.

BRIEF DESCRIPTION OF DRAWINGS

The figures are not necessarily to scale, emphasis instead generallybeing placed upon illustrative principles. The figures are to beconsidered illustrative in all aspects and are not intended to limit theinvention, the scope of which is defined only by the claims.

FIG. 1. SentiMag® magnetometer measurements (Abs Unit) at the lymphgland for various salt-based hypoosmotic solutions. Results presented asmean±SEM from 0 to 2 h, n=3.

FIG. 2. SentiMag® magnetometer measurements (Abs Unit) at the lymph nodefor various salt- and non-salt-based hypoosmotic solutions. Resultspresented as mean ±SEM from 0 to 120 min, n=3.

DETAILED DESCRIPTION

The invention relates in part to the discovery of compositions usefulfor rapid detection of lymph nodes in patients. These compositionsinclude suspensions of magnetic particles in a hypoosmotic solution. Theosmolality of the hypoosmotic solutions facilitates rapid drainage ortransport of the magnetic particles through the lymphatic system afterinjection, thereby reducing downtime between initial injection and lymphnode detection. Lymph nodes adjacent the injection site can be detectedrobustly in as little as 5 to 15 minutes after initial injection, whichis at least 50% faster than current methods, thereby permitting moreefficient pre-operative examination. In addition, the hypoosmoticsolutions of the invention are versatile solvents and can be used with awider range of excipients than isotonic or hypertonic solutions.Furthermore, rapid movement to the lymph nodes may reduce residualmarking, or tattooing, at the site of injection.

Hypoosmotic solutions within the meaning of the invention are aqueoussolutions having an osmolality of about 80 mOsm to about 160 mOsm.Isotonic solutions have an osmolaltiy of around 300 mOsm, andHyperosmotic solutions have an osmolaltiy of greater than 350 mOsm.

In preferred embodiments, an inorganic salt (e.g., sodium chloride) or aglycol (e.g., propylene glycol) is used to create the hypoosmoticsolution. Inorganic salt solutions (e.g., sodium chloride) having about0.01% w/v to about 0.6% w/v of a salt yield suitable hypoosomoticsolutions for use with the invention. Glycol solutions (e.g., propyleneglycol) having about 0.5% w/v to about 1.5% w/v of a glycol yieldsuitable hypoosomotic solutions for use with the invention.

Hypoosmotic solutions can be made using suitable inorganic saltsincluding, for example, monovalent and divalent salts such as sodiumchloride, potassium chloride, magnesium chloride, ammonium chloride,sodium bicarbonate, sodium bisulfate, sodium sulfate, ammonium sulfate,sodium phosphate, potassium phosphate, calcium chloride, magnesiumsulfate, potassium acetate, and sodium acetate.

Hypoosmotic solutions can be made using suitable glycols including, forexample, short chain, linear or branched alkyl glycols, such aspropylene glycol.

The magnetic particles can be composed of a suitable magnetic materialand one or more coatings. In some embodiments, the magnetic particlescontain an iron oxide such as magnetite and/or maghemite. The magneticcore can be surrounded by a biocompatible coating to reduce toxicity,prevent agglomeration of the particles, or to modify residence time inthe body. Suitable coatings include, for example, dextran,carboxydextran, other sugars, albumin, polyethylene glycol (PEG),biocompatible polymers, pegylated starch, polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), polyethyleneimine (PEI), polyglucosesorbitol carboxymethylether and chitosan. Other coating materialsinclude metals such as gold, pegylated colloidal gold nanoparticles,silver, carbon, silica, silicones, aminosilanes and ceramics. To exhibitsuperparamagnetic behavior, the magnetic cores of the particles shouldbe below a certain diameter, typically in the range 3-25 nm, dependingon the material and structure.

Magnetic particles can also be functionalized to allow them to localizein particular tissue or cell types, for example cancerous cells, or totarget particular biological systems in order to deliver therapies tothose areas. Functionalization is achieved by attaching or coating withbiovectors comprising, for example, antibodies, enzymes or proteins.

In one embodiment, iron oxide is used as the magnetic core because ofits low toxicity, but other materials that can form a superparamagneticcore also are acceptable. The core material should be capable of beingmagnetically ordered. It may be a metal, such as cobalt, iron, ornickel, a metal alloy, rare earth and transition metal alloy, M-type orspinel ferrite containing aluminium, barium, bismuth, cerium, chromium,cobalt, copper, dysprosium, erbium, europium, gadolinium, holmium, iron,lanthanum, lutetium, manganese, molybdenum, neodymium, nickel, niobium,palladium, platinum, praseodymium, promethium, samarium, strontium,terbium, thulium, titanium, vanadium, ytterbium, and yttrium or amixture thereof. The core can also be formed by oxidising a combinationof an iron(II) salt and another metal salt. The metal salts which arebeneficial include salts of aluminium, barium, bismuth, cerium,chromium, cobalt, copper, dysprosium, erbium, europium, gadolinium,holmium, iron, lanthanum, lutetium, manganese, molybdenum, neodymium,nickel, niobium, palladium, platinum, praseodymium, promethium,samarium, strontium, terbium, thulium, titanium, vanadium, ytterbium,and yttrium.

The osmolality of the hypoosmotic solutions have the further advantageof permitting combination with a wide range of excipients, resulting indiverse formulation options. Suitable excipients that can be used withthe hypoosmotic solutions of the invention include, for example:

-   -   co-solvents such as ethanol, propylene glycol, polypropylene        glycol, PEG 400, glycerol, benzyl alcohol, and combinations        thereof;    -   oils such as lipids, liquid paraffin, sesame oil, PEG vegetable        oil, and combinations thereof; surfactants such as polyoxylene        fatty acid esters, polyoxyl 40 castor oil, polysorbate 20,        polysorbate 80, and combinations thereof;    -   liposomes such as lecithin, egg lecithin, phosphatidyl glycerol,        phospholipid, egg phospholipid, and combinations thereof;    -   carbohydrates such as dextrose;    -   amino acids or amino acid mixtures, such as Aminosyn® II,        Travasol®, and HepatAmine®;    -   thickening/stabilizing agents such as carboxymethylcellulose;        and    -   buffers suitable for injection.        If an excipient increases the osmolality of a solution, the        amount of inorganic salt and/or glycol can be adjusted such that        the total osmolality of the hypoosmotic solution is between        about 80 mOsm and about 160 mOsm.

The compositions of the invention can be used to detect lymph nodes inhumans or any other mammal, such as pigs. For example, a hypoosmoticsolution comprising magnetic particles can be injected into a breastcancer patient. Magnetic particles in the solution are then detectedusing a magnetometer such as SentiMag® (Endomagnetics; Cambridge, U.K.)to reveal the location of the sentinel lymph nodes in the patient.

A further application of the hypoosmotic solution is in magnetichyperthermia where the solution is administered to the body for thepurpose of heating tissue. In this application the concentration ofnanoparticles is between 20 and 200 mg/ml and more preferably between100 and 140 mg/ml.

The hypoosmotic compositions of the invention can be suppliedready-to-use as part of a kit comprising a container, such as vial orsyringe, and instructions for administering the compositions.

EXAMPLES Example 1

Clinical trials with human patients using 2 ml Sienna+® (Endomagnetics;Cambridge, U.K.) was shown to give slow uptake in the axillary lymphnodes, with a poor external signal after 30 minutes. Sienna+® is highlyhypotonic, with an osmolality of ˜30 mOsm/kg. It was speculated thatwhen Sienna+® was injected into interstitial tissue, the surroundingcells rapidly absorbed water from the injection to maintain osmoticpressure. This would leave a more concentrated mass of Sienna+® whilesimultaneously reducing interstitial pressure, effectively reducingtransport to the lymph system. It is believed that an increase in volumeincreases interstitial pressure and thereby increases the speed ofuptake by the lymphatic system. However, large increases in volume mightprove uncomfortable to the patient. In addition, some potentialapplications for sentinel lymph node biopsy (e.g., bowel, melanoma, somehead and neck cancers) will not allow an increase in injection volume.It was hypothesized that an increased osmolality solution would providea quicker response as the fluid volume and pressure in the interstitialfluid would be maintained (isotonic) or even increased (for hypertonicinjection, where surrounding cells would expel water), thus increasingflow to the lymph nodes.

Methods

Pig mammaries were used as an in vivo lymph node model. An investigationwas performed to assess the effects of concentration and volume ofcarboxydextran coated maghemite nanoparticle solution on thebio-distribution of superparamagnetic iron oxide particles in pigs,following an injection of the solution directly into the 3^(rd) inguinalpapillar. The maghemite core had a diameter of about 5 nm, and thecarboxydextran coating increased particle diameter to about 60-70 nm.The aim of this study was to assess the bio-distribution of thesuperparamagnetic iron oxide particles in pig lymph nodes followinginjections of the maghemite nanoparticle solution prepared with 0.3, 0.6and 0.9% w/v sodium chloride. The influence of tonicity upon lymph nodebio-distribution of the particles was evaluated through use of aSentiMag® magnetic probe.

Prior to injection, pigs were sedated with an intramuscular combinationof azaperone and ketamine, followed by general anesthesia withintravenous sodium thiopental. Before administration, the administrationareas were washed and demarked.

All injections were made directly into the base of the 3rd inguinalpapilla. Each pig received a different injection in the left papilla andthe right papilla. Each of the test solutions was injected into threepapilla of different pigs (n=3). Table 1 shows the tested formulations.In Table 1, the “System” column corresponds to the curves in FIG. 1.

TABLE 1 Formulations tested. Total Iron System Tonicity Injection Volumeiron Concentration a 0.3% w/v saline 0.4 mL 10.4 mg 26 mg/mL b 0.6% w/vsaline 0.4 mL 10.4 mg 26 mg/mL c 0.9% w/v saline 0.4 mL 10.4 mg 26 mg/mLd 0.9% w/v saline 0.8 mL 20.8 mg 26 mg/mL e 0 0.2 mL 10.4 mg 52 mg/mL f0 0.4 mL 20.8 mg 52 mg/mL g 0 0.4 mL 10.4 mg 26 mg/mL (Sienna + ®,control) h 0 0.8 mL 10.4 mg 13 mg/mL

Carboxydextran coated maghemite nanoparticle solutions were prepared inwater. NaCl was added to the maghemite solution to the appropriateconcentration. For example, a 0.3% salt magnetic particle suspension wascreated by adding 0.3 mg of NaCl to a prediluted maghemite nanoparticlesolution. Sienna+® (˜26 mg/mL maghemite, 0.4 mL dose; system g) servedas the control.

Multiple readings were taken for each pig using an SentiMag® device, asdetailed in Table 2. Following the 72 h readings, the site of papillaand lymph nodes were removed from all animals for histological analysis.The results in FIG. 1 are averages (n=3) of the measurements taken atthe lymph nodes.

TABLE 2 SentiMag ® measurement sites and time points. After injectionMeasurement Prior to 30 2 5 15 30 1 2 6 24 72 location injection s minmin min min h h h h h Directly on papillar x x x x x x x x x x x 2ndinguinal papillar x x x x x x x x x x x Lymph node x x x x x x x x x x x

Results

Sienna+® in 0.3% w/v saline solution (FIG. 1, system a) was found to beas efficacious as both the 0.6% and 0.9% saline solutions (FIG. 1,systems b and c, respectively). This was surprising because a 0.3% w/vsolution is hypotonic (156 mOsm) compared to 0.6% w/v (270mOsm—approximately isotonic) and 0.9% w/v (384 mOsm—hypertonic). Suchstrong results for a 0.3% w/v solution are unexpected. It is believedthat the low tonicity will extend the range of formulation additives(excipients) that can be used, as compared to 0.6% and 0.9% solutions.

Specifically, increasing tonicity results in significantly more rapidtransport of iron particles through the lymphatic system. At 5 minutespost injection, the addition of 0.3%, 0.6%, and 0.9% salt to Sienna+®(FIG. 1, systems a, b, and c, respectively) result in a five-foldincrease in signal measured at the lymph gland as compared to thecontrol, Sienna+® (FIG. 1, system g). As a result, a treating physicianneed only wait 5-15 minutes before beginning a procedure, which reducesthe wait time by at least 50% as compared to Sienna+® alone.

Furthermore, at the 30 minute time point, the impact of adding 0.3%,0.6%, or 0.9% w/v sodium chloride to Sienna+® (FIG. 1, systems a, b andc, respectively) is equivalent to doubling the concentration of iron(FIG. 1, system f). Consequently, increasing tonicity requires lesstotal iron be used per injection, thereby reducing costs and sideeffects.

Thus, a hypoosmotic <0.6% NaCl solution and more preferably <0.3% NaClsolution provides the same rapid transport to the lymph nodes as anisotonic (e.g. 0.6% NaCl) or even a hypotonic (e.g. 0.9% NaCl) solutionbut without requiring such a large amount of salt to be included in thesolution.

A more hypotonic solution containing 0.05% w/v NaCl showed nosignificant improvement over Sienna+®. The “trigger-point” for tonicitybenefit is therefore somewhere between about 80 mOsm and about 156 mOsm.Thus, a 0.05% to 0.3% NaCl solution or preferably a 0.1% to 0.3% NaClsolution, or more preferably a 0.2% to 0.3% NaCl solution exhibits bothrapid uptake and versatility as an excipient.

Example 2

Similar in vivo pig studies were undertaken to investigate hypoosmoticsolutions comprising alternative solutes. All injections were madedirectly into the base of the 3rd inguinal papilla. Each pig received adifferent injection in the left papilla and the right papilla. Each testsolution was injected into the three papilla of different pigs (n=3).Table 3 shows the tested formulations. In Table 3, the “System” columncorresponds to the curves in FIG. 2.

TABLE 3 Solution formulations. Tonicity System Solution (mOsm/kg) 1Sienna + with 0.3% NaCl and 0.1% HA 132 3 Sienna + with 0.3% NaCl and 1%polysorbate 20 135 5 Sienna + with 0.5% propylene glycol 128 6 Sienna +with 0.3% NaCl (control) 126 7 Sienna + 0.75% glycerol 136 13 Sienna +0.5% propylene glycol and 1.8% glycerol 297

System 6, Sienna+® with 0.3% NaCL served as the control. Multiplereadings were taken for each pig using a SentiMag® device, as detailedin Table 4. The results in FIG. 2 are averages (n=3) of the measurementstaken at the lymph nodes.

TABLE 4 SentiMag ® measurement sites and time points. Measurement Priorto After injection location injection 30 s 2 min 5 min 15 min 30 min 1 h2 h 72 h Injection site x x x x x x x x x 2nd inguinal papillar x x x xx x x x x Inguinal lymph node x x x x x x x x x

As shown in FIG. 2, system 6 (Sienna+0.3% NaCl—CONTROL) resulted in thefastest delivery to the lymph gland. Salt therefore appears to be thebest potentiator for delivery. 0.5% polyethylene glycol (system 5) alsoappears to be efficacious, resulting in rapid delivery to the lymphgland within 5-15 minutes of injection. Accordingly, in someembodiments, glycols can be used as a solute to create a hypoosomoticsolution comprising magnetic particles.

The polysorbate (system 3) and glycerol (system 7) formulations were thepoorest performing formulations over the first two hours, despite thetonicity being equal to that of the NaCl control (system 6), indicatingthat polysorbate and glycerol potentially inhibit delivery to the lymphglands. Similarly, addition of hyaluronic acid (MW 108,000 Daltons)appears to retard delivery when combined with 0.3% NaCl.

It should be noted that the method of administration of the solutionwill depend on the particular site in the body at which it is beingadministered. For sentinel lymph node biopsy, the injection may beinterstitial, sub-cutaneous, intradermal or intramuscular. For magnetichyperthermia, the solution may be administered by any of these injectionmethods or via a catheter or infusion into a region of tissue, a bodycavity, or vessel.

It should be understood that the order of steps or order for performingcertain actions is immaterial, provided that the invention remainsoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

Where a range or list of values is provided, each intervening valuebetween the upper and lower limits of that range or list of values isindividually contemplated and is encompassed within the invention as ifeach value were specifically enumerated herein. In addition, smallerranges between and including the upper and lower limits of a given rangeare contemplated and encompassed within the invention. The listing ofexemplary values or ranges is not a disclaimer of other values or rangesbetween and including the upper and lower limits of a given range.

What is claimed is:
 1. A hypoosmotic suspension for medical injection comprising: about 13 mg/mL to about 200 mg/mL of superparamagnetic particles; and an osmolyte selected from either about 0.01% w/v to about 0.6% w/v of an inorganic salt or about 0.5% w/v to about 1.5% w/v of a glycol.
 2. The hypoosmotic suspension of claim 1, wherein the superparamagnetic particles are iron oxide.
 3. The hypoosmotic suspension of claim 1, comprising about 13 mg/mL of superparamagnetic particles.
 4. The hypoosmotic suspension of claim 1, further comprising an excipient.
 5. The hypoosmotic suspension of claim 1 wherein the superparamagnetic particles are coated.
 6. The hypoosmotic suspension of claim 5, wherein the coating comprises dextran. The hypoosmotic suspension of claim 1, wherein the inorganic salt is sodium chloride.
 8. The hypoosmotic suspension of claim 1, wherein the suspension is used for the detection of sentinel nodes and comprises about 0.05%-0.3% w/v of the inorganic salt.
 9. The hypoosmotic suspension of claim 1, wherein the glycol is propylene glycol.
 10. The hypoosmotic suspension of claim 1, wherein the suspension is used for magnetic hyperthermia treatment and comprises about 20 mg/ml-200 mg/ml of the superparamagnetic particles.
 11. A method of locating a lymph node in a patient, the method comprising the steps of: providing the hypoosmotic suspension of claim 1; injecting the hypoosmotic suspension into the patient; waiting until the superparamagnetic particles become entrapped in a lymph node; and detecting the location of the lymph node by detecting the location of the superparamagnetic particles.
 12. A method of locating a lymph node in a patient, the method comprising the steps of: providing a hypoosmotic suspension of claim 1 comprising superparamagnetic particles; injecting the hypoosmotic suspension into the patient; and detecting a lymph node within 10 minutes of injection by detecting the location of the superparamagnetic particles, the detecting sufficient to immediately begin a medical procedure on the lymph node based on the detecting.
 13. A method of treating a patient using magnetic hyperthermia, the method comprising the steps of: providing the hypoosmotic suspension of claim 1; injecting the hypoosmotic suspension into the patient; and exposing the patient to an alternating magnetic field.
 14. The hypoosmotic suspension of claim 1, wherein the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, ammonium chloride, sodium sulfate, sodium phosphate, potassium phosphate, calcium chloride, magnesium sulfate, potassium acetate, and sodium acetate. 